Binding assays generally have a range of analyte concentrations, the dynamic range, for which the reported signal is dependent upon the amount of analyte present. Where the amount of analyte exceeds the dynamic range of the assay, saturation of binding sites occurs and the reported signal is not indicative of the true analyte concentration. Likewise, when the amount of analyte present in the sample falls below the lower threshold of the assay's dynamic range, the assay is insufficiently sensitive to the actual analyte concentration, and the reported signal will also not be indicative of the true analyte concentration.
Two approaches have conventionally been employed in single measurement assays for analytes having concentrations above the upper limit of the dynamic range of the assay: (1) diluting the sample to reduce the concentration to within the assay range; and (2) reducing the binding reaction time to prevent saturation of binding sites. In the first approach, multiple dilutions of a sample are sometimes made and individually tested so as to increase the likelihood that one of the dilutions is within the dynamic range of the assay. In the second approach, multiple experiments can be carried out using different binding reaction times so as to increase the likelihood that one of the experiments has a binding reaction time that produces an assay signal within the dynamic range of the assay.
It is increasingly desirable to assay multiple different analytes simultaneously in the same sample. Multiplexing permits greater throughput, minimizes sample volume and handling, provides internal standardization control, decreases assay cost and increases the amount of information that is obtainable from each sample. Various approaches for conducting multiplexed assays have been described. For example, multiplexed testing is described in U.S. patent application Ser. Nos. 10/185,274 and 10/185,363, both filed on Jun. 28, 2002, entitled “Assay Plates, Reader Systems and Methods For Luminescence Test Measurements,” published as U.S. Publ. No. 20040022677, U.S. patent application Ser. No. 10/238,960, filed Sep. 10, 2002, entitled “Methods, Reagents, Kits and Apparatus for Protein Function,” published as US Pat. Pub. No. 20030207290, U.S. patent application Ser. No. 10/238,391, filed Sep. 10, 2002, entitled “Methods and apparatus for conducting multiple measurements on a sample”; published as US Pat. Publ. No. 20030113713, Provisional U.S. Patent Application No. 60/517,606, filed on Nov. 4, 2003, entitled “Modular Assay Plates, Reader System and Methods For Test Measurements”; and U.S. patent application Ser. No. 10/744,726, filed on Dec. 23, 2003, entitled “Assay Cartridges and Methods of Using Same”, each of which is incorporated by this reference. One approach to multiplexing binding assays involves the use of patterned arrays of binding reagents (see, e.g., U.S. Pat. Nos. 5,807,522 and 6,110,426, both entitled “Methods for Fabricating Microarrays of Biological Samples” issued Sep. 15, 1998 and Aug. 29, 2000 respectively, Delehanty J B, Printing functional protein microarrays using piezoelectric capillaries, Methods Mol Biol. (2004) 278:135-44; Lue R Y, Chen G Y, Zhu Q, Lesaicherre M L, Yao S Q, Site-specific immobilization of biotinylated proteins for protein microarray analysis, Methods Mol Biol. (2004) 278:85-100; Lovett, Toxicogenomics: Toxicologists Brace for Genomics Revolution, Science (2000) 289: 536-537; Berns A., Cancer: Gene expression in diagnosis, Nature (2000) 403, 491-492; Walt, Molecular Biology: Bead-based Fiber-Optic Arrays, Science (2000) 287: 451-452. Another approach involves the use of binding reagents coated on beads that can be individually identified and interrogated. International Patent publication WO9926067A1 (Watkins et al.) describes the use of magnetic particles that vary in size to assay multiple analytes; particles belonging to different distinct size ranges are used to assay for different analytes. The particles are designed to be distinguished and individually interrogated by flow cytometry. Vignali has described a multiplex binding assay in which 64 different bead sets of microparticles are employed, each having a uniform and distinct proportion of two dyes (Vignali, D. A. A., “Multiplexed Particle-Based Flow Cytometric Assays,” J. Immunol. Meth. (2000) 243:243-255). A similar approach involving a set of 15 different beads of differing size and fluorescence has been disclosed as useful for simultaneous typing of multiple pneumococcal serotypes (Park, M. K. et al., “A Latex Bead-Based Flow Cytometric Immunoassay Capable Of Simultaneous Typing Of Multiple Pneumococcal Serotypes (Multibead Assay),” Clin Diagn Lab Immunol. (2000) 7:486-9). Bishop, J. E. et al. have described a multiplex sandwich assay for simultaneous quantification of six human cytokines (Bishop, J. E. et al., “Simultaneous Quantification of Six Human Cytokines in a Single Sample Using Microparticle-based Flow Cytometric Technology,” Clin Chem. (1999) 45:1693-1694).
A significant complexity arises in conducting multiplexed assays, however, from the fact that the individual analytes may occur in widely different abundance. As a consequence, it may not always be possible to find a single set of conditions (e.g., sample dilution or binding reaction time) which bring all analytes of interest within the assay's dynamic range. An appropriate dilution level for a high concentration analyte, for example, may leave the low concentration analyte undetectable.